Optimization of -Mediated Transformation of EL000614, an Endolichenic Fungus Producing Grammicin.

An endolichenic fungus EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an -mediated transformation (ATMT) protocol for to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of with strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase () gene and the gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of .